CLCA2 is a mammary epithelial cell surface protein that is induced in response to contact inhibition and cellular stress. Its expression is downregulated in breast tumors, most dramatically in invasive and metastatic tumors and cell lines. Restored expression inhibits growth, migration, and invasion of metastatic breast carcinoma lines. Originally thought to be a multipass chloride channel, the protein was recently shown to be almost entirely extracellular and to undergo a cleavage that releases the 100 kDa ectodomain into the extracellular space. Our preliminary data indicate that the soluble ectodomain (sCLCA2) is preferentially cytotoxic to triple-negative breast carcinoma cells. This type of breast cancer lacks targets for antagonists of estrogen and growth factor receptors that have driven the increase in overall survival of patients with ER+ and HER2+ breast cancers. Triple-negative breast cancers are typically invasive, metastatic, chemotherapy-resistant and prone to relapse, and are therefore associated with the poorest outcome. A low-toxicity agent is needed that can suppress growth of both primary tumors and metastatic lesions derived from such cells. As an endogenous human negative regulator of growth and migration, sCLCA2 might be safely administered over an extended period either prior to or following surgical removal of the primary tumor. Unlike humanized chimeric monoclonal antibodies, development and production costs of sCLCA2 should be low and the path to FDA approval straightforward. Our specific aims are: 1.To determine whether sCLCA2 applied as monotherapy or in combination with chemotherapeutic drugs can specifically stimulate apoptosis in breast tumor cells but spare normal cells. 2. To delineate the tumoricidal domain of sCLCA2 and identify its receptor or target on the tumor cell. 3. To determine whether sCLCA2 or its subdomain can suppress growth and metastasis of orthotopic mouse xenografts or spontaneous breast tumors without toxicity to normal tissues. Our long-term goal is to determine the molecular mechanism(s) by which sCLCA2 antagonizes tumor progression and limits the proliferation of normal epithelial cells. The proposed studies are expected to reveal such pathways and potentially produce a novel, low-toxicity biotherapeutic for treatment of both primary and metastatic breast cancer. PUBLIC HEALTH RELEVANCE: This project capitalizes on an exciting observation that we made only this summer, that the secreted form of a cell surface protein CLCA2 is preferentially toxic to breast tumor cells with a triple-negative phenotype or basaloid phenotype. We measure its stability, toxicity to normal tissues and efficacy against tumor cells in culture and in an animal model. We determine how it causes tumor cell death. We determine the structural feature responsible and its target on the tumor cell.